A single amino acid tunes Ca2+ inhibition of brain liver intestine Na+ channel (BLINaC)

Abstract

Ion channels of the degenerin/epithelial Na(+) channel gene family are Na(+) channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na(+) channel (BLINaC) is an ion channel of the degenerin/epithelial Na(+) channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca(2+). Removal of Ca(2+) leads to robust currents and increases Na(+) selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca(2+) affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca(2+). In addition, mBLINaC is more selective for Na(+) and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na(+) channel.

FIGURE 1.

Rat BLINaC is inhibited by physiological concentrations of extracellular Ca²⁺. A, representative current trace of oocytes expressing rBLINaC. Amiloride-sensitive (4 mm) current was recorded in 1.8 mm and 10 nm [Ca²⁺]_e, respectively; holding potential was −70 mV. B, upper, representative current trace recorded in the presence of decreasing [Ca²⁺]_e. B, lower, concentration-dependent inhibition of rat BLINaC by [Ca²⁺]_e. Currents were normalized to the current in the presence of 10 nm [Ca²⁺]_e; n = 10. The dashed line highlights a Ca²⁺ concentration of 1.8 mm. Error bars, S.E. C, upper, representative current trace recorded in the presence of increasing concentrations of amiloride (Amil); [Ca²⁺]_e was 1.8 mm. C, lower, concentration-dependent inhibition of rBLINaC by amiloride in physiological [Ca²⁺]_e (filled squares, n = 10) and low [Ca²⁺]_e (open squares, n = 8); currents were normalized to the current in the absence of amiloride.

FIGURE 2.

Mouse BLINaC is constitutively open. A, comparison of amiloride-sensitive current amplitudes of oocytes expressing rBLINaC (undiluted cRNA) or mBLINaC (25-fold diluted cRNA), n = 8, **, p < 0.005. B, representative current trace from an oocyte injected with 100-fold diluted mBLINaC. Amiloride-sensitive (100 μm) current was recorded in 1.8 mm and 10 nm [Ca²⁺]_e, respectively. Holding potential was −70 mV. C, concentration-dependent inhibition of mBLINaC by [Ca²⁺]_e. Currents were normalized to the current in the presence of 10 μm [Ca²⁺]_e; n = 16. D, concentration-dependent inhibition of mBLINaC by amiloride; n = 10. Currents were normalized to the current in the absence of amiloride.

FIGURE 3.

A single amino acid is responsible for the species difference between rat and mouse BLINaC. A, left, schematic drawings of rat and mouse BLINaC and chimeras. A, right, amiloride-sensitive current amplitudes of rat and mouse BLINaC and chimeras. 100 μm amiloride was used for mBLINaC and chimera loop2, 4 mm amiloride for all others. cRNA was injected undiluted except for mBLINaC and loop2, which were injected 25-fold diluted. Results represent data from 1 week, n = 5. **, p < 0.005. Error bars, S.E. B, upper, alignment of the amino acid sequences of the second part of the ECD of rat and mouse BLINaC. Amino acids different between rat and mouse are marked by arrows. B, lower, amiloride-sensitive current amplitudes of oocytes expressing rBLINaC carrying individual amino acid substitutions. 100 μm amiloride was used for mBLINaC and rBLINaC-A387S, 4 mm for all others. All cRNAs were injected undiluted except for mBLINaC and rBLINaC-A387S, which were injected 25-fold diluted. Results represent data from 1 week, n = 8. **, p < 0.005.

FIGURE 4.

Amino acid 387 determines Ca²⁺ affinity and amiloride affinity of BLINaC. A, concentration-dependent inhibition of rBLINaC-A387S by [Ca²⁺]_e (black triangles). Currents were normalized to the current in the presence of 10 μm Ca²⁺_e, n = 11. Dose-response curves of rBLINaC (gray squares) and mBLINaC (gray circles) from Figs. 1B and 2C, respectively, are shown for comparison. B, concentration-dependent inhibition of rBLINaC-A387S by amiloride (black triangles). Currents were normalized to the current in the absence of amiloride, n = 14. Dose-response curves of rBLINaC (gray squares) and mBLINaC (gray circles) from Figs. 1C and 2D, respectively, are shown for comparison.

FIGURE 5.

Amino acid 387 determines ion selectivity of BLINaC. A, normalized mean current-voltage relationships of amiloride-sensitive currents of rBLINaC (black squares), mBLINaC (black circles), and rBLINaC-A387S (gray triangles), determined in 1.8 mm [Ca²⁺]_e. For rBLINaC, I-V relationships determined in 10 nm [Ca²⁺]_e are also shown (gray squares). The holding potential was increased stepwise from −120 to +60 mV (20- or 30-mV steps) in the absence and the presence of amiloride (100 μm for mBLINaC and rBLINaC-A387S, 4 mm for rBLINaC). Currents in the presence of amiloride were subtracted from currents in the absence of amiloride to yield the amiloride-sensitive currents at each holding potential. B, left, representative current traces for rBLINaC, mBLINaC, and rBLINaC-A387S. Currents were recorded at alternating holding potentials of −70 and +50 mV, in 1.8 mm [Ca²⁺]_e and 140 mm varying extracellular monovalent cations. B, right, conductances of mBLINaC, rBLINaC, and rBLINaC-A387S. Conductance was calculated according to the following equation: g = ([I_{−70 mV}]+[I_{+50 mV}])/120 mV. Conductances in the presence of Li⁺ and K⁺ were normalized to the conductance in the presence of Na⁺. rBLINaC, n = 8; mBLINaC, n = 8; rBLINaC-A387S, n = 11; **, p < 0.005. C, left, representative current trace for rBLINaC in 1.8 mm and 10 nm [Ca²⁺]_e. Holding potential was alternated between −70 and +50 mV. C, right, normalized conductances in 1.8 mm and 10 nm [Ca²⁺]_e; n = 9; **, p < 0.005.

FIGURE 6.

BLINaC is proton-permeable. A, representative current traces of an oocyte expressing mBLINaC. Left, bath solutions with varying proton concentrations were applied and exchanged every 10 s in the presence of Na⁺. Right, after replacement of Na⁺ by NMDG⁺, increasing concentrations of protons were applied. The part of the current trace marked by the box is magnified at the bottom. B, representative current-voltage relationship of mouse BLINaC obtained in 1 mm Na⁺ and with two different proton concentrations (100 nm corresponding to pH 7, black curve, and 100 μm corresponding to pH 4, gray curve). Voltage was ramped from −120 to +60 mV in 9 s.